Proc. Nati. Acad. Sci. USAVol. 89, pp. 8522-8526, September 1992Immunology

Heterogeneity of immunoglobulin-associated molecules on human Bcells identified by monoclonal antibodiesTETSUYA NAKAMURA*, HIRoMI KUBAGAWA*, AND MAx D. COOPER*t*Division of Developmental and Clinical Immunology, Departments of Medicine, Pathology, Pediatrics, and Microbiology, University of Alabama atBirmingam, and tHoward Hughes Medical Institute, Birmingham, AL 35294

Contributed by Max D. Cooper, May 26, 1992

ABSTRACT Two covalently linked transmembrane mol-ecules, encoded in mice by the mb-i and B29 genes, have beendefined as integral components of the antibody receptor unitsexpressed on B cells. We have produced monoclonal antibodiesagainst an exposed extracellular epitope on the putative humanequivalent of the mouse B29 product. These antibodies, CB3-1and -2, were used to show that cytoplasmic expression of thismolecule begs in human pro-B cells (terminal deoxynude-otidyltransferase-poitive, , chain-negative), whereas surfaceexpression coincides strictly with surface immunolobulin ex-pression of all isotypes. Immunochemical analysis ofthe humanimmunoglobulin-associated molecules revealed greater molec-ular heterogeneity than has been noted for the murine ana-logues. This molecular heterogeneity of immunoglobulin-associated molecules varied as a function of differentiationstage and the immunoglobulin isotypes expressed by B-lineagecells. Our data support the hypothesis that biochemical heter-ogeneity of the surface immunoglobulin-assocated moleculesmay contribute to the variabllity in biological effects of antigenreceptor crossllnkage on B cells of different maturationalstages. Because the CB3 antibodies are capable of down-modulating the antigen receptors on all B cells, they may provetherapeutically useful as universal B-cell suppressants.

Signal transduction through cell surface immunoglobulin(sIg) receptors requires the presence ofphysically associatedmolecules, as the cytoplasmic tail of slg molecules is veryshort. Two molecules encoded by B-cell-specific genes, mb-i(1) and B29 (2), have been shown to be noncovalentlyassociated with sIgM and sIgD to form antigen receptor unitson B cells that resemble the CD3/T-cell-receptor complexes(3-10). The mb-i products are called a chains, and the B29products (3 chains (9).

Characteristic of the sIg-mediated signal transductionpathways is that the initial activation cascades result indifferent biological effects, depending on the B-cell matura-tion stage and the sIg isotypes that are crosslinked (11-13).These complicated effects of sIg crosslinkage are not yet fullyexplained since, with the exception of variation in molecularmass ofthe mb-i products due to differential glycosylation (5,14), the components of the B-cell antigen receptor complexappear identical regardless ofthe B-cell maturation stage andthe sIg isotype (1, 2, 6, 7).

In the present experiments, IgM-associated molecules onhuman B cells were isolated and used to produce monoclonalantibodies (mAbs) against exposed determinants on the hu-man Ig-associated molecules. These mAbs were employed toinvestigate the antigen receptor units on B cells at variousstages of maturation and to begin to explore their therapeuticpotential.- Here we describe the distribution and biochemicalfeatures of the Ig-associated molecules, using two mAbs thatappear to recognize extracellular epitopes of the human B29

counterpart and induce down-modulation of the antigenreceptors on B cells.

MATERIALS AND METHODSAntibodies and Cells. Anti-human ,u chain (SA-DA44),

anti-human 8 (SIA6-2), anti-human K (TB28-2), anti-human A(1-155-2) and anti-mouse K (187-1) mAbs were describedpreviously (15, 16). Anti-CD19, -CD20, and -CD38 mAbswere purchased from Becton Dickinson. Affinity-purifiedgoat antibodies specific for mouse or human Ig, and theappropriate developing reagents, were from Southern Bio-technology Associates (Birmingham, AL). Pre-B cell lines207 (Ig-) and 697 (cytoplasmic and surface iz+, surrogate lightchain+), B cell lines Ramos (surface ,uA+), Daudi (surface,UK+), and IM9 (surface 'ylK+), and a T cell line, MOLT4,were described previously (17).

Preparation of mAbs Specific for Ig-Assocated Molecules.Ramos B cells (4 x 109) were lysed in 1% digitonin (Aldrich)lysis buffer and centrifuged for 30 min at 17,000 X g. Thesoluble components were incubated with Sepharose 4B co-pled with the SA-DA4-4 mAb and, after extensive washing,bound proteins were eluted by either Laemmli's samplebuffer or 1% Nonidet P-40 (NP-40) lysis buffer. Proteinseluted with sample buffer were subjected to SDS/PAGEunder reducing conditions and then stained with Coomassiebrilliant blue. Bands corresponding to 36-52 kDa, shown tocontain Ig-associated molecules in pilot experiments, wereexcised and used for the three initial immunizations ofBALB/c mice. Proteins eluted with 1% NP40 lysis bufferwere injected in two additional immunizations. Finally, 1 dayafter a booster immunization with viable Ramos cells (2 x107), lymph node cells were harvested and fused with P3-X63-Ag8.653 myeloma variant cells. Culture supernatants ofhybridomas were tested in flat-bottomed 96-well platescoated first with goat anti-human ,u antibody and then with a1% digitonin lysate of Daudi cells as a source of IgM-associated molecules (5 x 105 cells per well). Bound anti-bodies were detected with (3-galactosidase-labeled goat anti-mouse Ig antibody, followed by o-nitrophenyl 3-D-galacto-pyranoside. Each supernatant was also tested for ELISAreactivity with an IgM paraprotein.

Immunofluorescence Analyses. Cells (5 x 105) were incu-bated with antibodies (50 pg/ml) for 20 min at 40C unlessotherwise indicated. For single-color surface staining cellswere incubated with purified CB3-1, CB3-2, SA-DA44, orTB28-2, followed by a biotinylated goat anti-mouse Ig anti-body and then phycoerythrin-conjugated streptavidin (10

lg/ml). For two- or three-color staining, cells were incubatedwith biotinylated CB3-1, developed with phycoerythrin-conjugated streptavidin and then stained with fluorescein-labeled anti-CD19 or anti-CD38 mAb or a mixture of fluo-

Abbreviations: sIg, surface immunoglobulin; mAb, monoclonal an-tibody; TdT, terminal deoxynucleotidyltransferase; NP-40, NonidetP-40.

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rescein-labeled goat anti-human ,u antibody and peridininchlorophyll protein-labeled anti-CD19 mAb. For intracyto-plasmic staining, unstained cells or cells stained with arhodamine-labeled goat anti-human ,u antibody were centri-fuged onto glass slides, fixed with 5% acetic acid/95%ethanol, and then stained with appropriate antibody prepa-rations. For nuclear staining of terminal deoxynucleotidyl-transferase (TdT), cells fixed in 100% methanol were incu-bated with rabbit anti-TdT antibody (Supertechs, Bethesda,MD) and then with fluorescein-labeled goat anti-rabbit Igantibody. Counterstains were with biotinylated CB3-1 orSA-DA4-4 antibodies, followed by rhodamine-conjugatedstreptavidin.Modulation Assay. Blood mononuclear cells were cultured

with CB3-1, CB3-2, or mixture of goat anti-human K andanti-human A antibodies (50 u.g/ml) for 16 hr at 37°C. Cellswere then washed and stained with CB3-1 or CB3-2 plusfluorescein-labeled goat anti-mouse Ig antibody, fluorescein-labeled anti-CD20 mAb or fluorescein-labeled goat anti-human heavy chain antibodies. After incubation with normalmouse serum, the B cells were counterstained with phyco-erythrin-labeled anti-CD19 mAb and the mean fluorescenceintensity was determined for B-cell expression of each anti-gen with and without prior antibody modulation.

Immunoprecipitation. Cells surface labeled with Na125I(Amersham) were lysed in either 1% NP-40 or 1% digitoninlysis buffer containing 150 mM NaCl, 50 mM Tris HCl (pH7.4), 5 mM EDTA, 0.05% sodium azide, 1 mM diisopropylfluorophosphate, leupeptin (10 Mg/ml), pepstatin A (1 ,ug/ml), chymostatin (2 pg/ml), and 1 mM phenylmethylsulfonylfluoride. Cell lysates were first cleared with Sepharose 4Bcoupled with an isotype-matched antibody of irrelevant spec-ificity and then were incubated with Sepharose 4B coupledwith specific antibodies. Bound molecules were dissociatedwith Laemmli's buffer, resolved by SDS/l0o PAGE, andidentified by autoradiography. N-Glycanase (Genzyme)treatment of eluted proteins was done according to manu-facturer's instructions.

Immunoblotting. Digitonin lysates of Ramos, 697, andMOLT4 cells were incubated with Sepharose 4B coupledwith SA-DA4-4, and bound molecules were eluted with 1%NP-40 lysis buffer. Eluted molecules were resolved by SDS/PAGE and transferred to a nitrocellulose membrane. Afterblocking, the membrane was incubated with CB3-1 andCB3-2 and then with 187-1 labeled with 125I.

RESULTSCB3-1 and -2 mAbs Identify an Exposed Epitope on sIg-

Associated Molecules. Of the antibody products from >500hybridomas, 37 were reactive with the sIgM complex fromDaudi B cells but were nonreactive with secreted IgMmolecules. Two of these hybridomas were selected becauseof their reactivity with viable Ramos B cells and 697 pre-Bcells, subcloned by limiting dilution, and designated CB3-1and -2. Both mAbs were determined to be of mouse ylKisotype.Modulation studies revealed that incubation of normal B

cells with CB3-1 or CB3-2 led to down-modulation in expres-sion ofthe corresponding cell surface antigen and that the sIgmolecules were comodulated (Table 1). Conversely, anti-Igantibodies modulated both the sIg and CB3-1 antigens,whereas cell surface CD20 expression was unaffected bypretreatment with any of these antibodies. These resultsindicated the physical association of sIg molecules with theCB3 antigens on normal B cells.When cell surface proteins from Ramos B cells were lysed

with NP-40 detergent, the CB3-1 and -2 immunoprecipitatesmigrated on gel electrophoresis as a single broad band at82-95 kDa (Fig. lc). Under reducing conditions, three mol-

Table 1. Comodulation of slg and the CB3-1/CB3-2 antigen

Antibody % of control MFItreatment sIg CB3-1 CB3-2 CD20

Anti-K,+A 6.2 5.5 ND 87.1CB3-1 17.7 6.4 ND 89.9CB3-2 26.2 ND 14.0 94.0

Peripheral blood lymphocytes were cultured for 16 hr with goatanti-K+A, CB3-1, or CB3-2 antibodies, and percentage of the controlmean fluorescence intensity (MFI) was calculated for the indicatedantigens on CD19+ B cells. ND, not done.

ecules of45, 40, and 37 kDa (p45, p40, and p37 proteins) wereresolved from the CB3-1 and -2 precipitates (Fig. la),whereas an anti-human A antibody precipitated only the Auheavy chains and A light chains under the same reducingconditions. In contrast, when the cells were lysed with amilder detergent, digitonin, to avoid dissociation of nonco-valently linked molecules, the anti-human AL, CB3-1, andCB3-2 antibodies all precipitated the A heavy and A lightchains in addition to p45, p40, and p37 (Fig. lb). The p40 bandwas sometimes obscured by diffuse migration of overlappingp45 and p37 bands. These results indicate that both CB3antibodies recognize covalently linked dimers consisting ofp45, p40, and p37 molecules that are noncovalently associ-ated with sIgM molecules on B cells.To determine which of the associated proteins were rec-

ognized by the CB3-1 and -2 mAbs, we performed Westernblot analysis of Ramos B-cell and 697 pre-B-cell lysatestogether with MOLT4 T-cell lysates as a control. Both mAbsreacted with a prominent 37-kDa protein of Ramos B cellsand a 34-kDa protein of 697 pre-B cells (Fig. 2; see also Fig.Sc, lane 2) but were unreactive with MOLT4 T-cell constit-uents. A faint 34-kDa band was also recognized by bothmAbs on Ramos cells. Since the p45 and p40 IgM-associatedmolecules have been identified as the human mb-l-encodeda chains (18), these results suggest that the CB3 antibodiesrecognize human (-chain equivalents which exhibit sizeheterogeneity in representative pre-B and B cells.

Surface Expression of the CB3 Antigen During B-Cell Dif-ferentiation. Competition immunofluorescence assays re-vealed that binding of the CB3-1 antibody to Ramos cells was

a. NP-401 2 3 4

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antibodies. 1251-labeled Ramos cells were lysed in either NP-40 (a andc) or digitonin (b and d) lysis buffer and proteins were immunopre-cipitated with Sepharose 4B beads coupled with an isotype-matchedcontrol mAb (lanes 1), anti-,u mAb (SA-DA4-4) (lanes 2), CB3-1(lanes 3), or CB3-2 (lanes 4). Immunoprecipitates were analyzed bySDS/110o PAGE under reducing (a and b) or nonreducing (c and d)conditions. Relative molecular mass standards (kDa) are indicated.

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FIG. 2. Western blot analysis of CB3-1 and -2 antigens expressedby pre-B and IgM+ B cell lines. Ig-associated molecules purified fromRamos (lanes 1), 697 (lanes 2), and MOLT4 cells (lane 3) wereresolved by SDS/PAGE under reducing conditions, transferred ontoa nitrocellulose membrane, and incubated with CB3-1 (a) or CB3-2(b) mAb.

inhibited equally well by the CB3-1 and -2 antibodies and viceversa (data not shown), suggesting that both antibodiesrecognized the same or contiguous epitopes. Since bothmAbs perfomed identically in all assays, only the resultsobtained with the CB3-1 antibody are shown in the followingexperiments.The CB3 mAbs were reactive by surface immunofluores-

cence with all of the sIg+ cell lines tested, but with none ofthe sIg- cell lines examined, regardless of their lineage (datanot shown). Expression of the cell surface CB3 antigen on

pre-B and B cell lines paralleled sIg expression (Fig. 3). Thiswas true for both sIgM+ (Ramos) and sIgG1+ (IM9) cell lines.When peripheral blood cells were examined, the antigen wasfound only on the CD19+ B cells, all of which were CB3+(Fig. 4a). In contrast, terminally differentiated myeloma cellsdid not express the CB3 antigen (Fig. 4b).When bone marrow lymphocytes were examined, only

40%o of the CD19+ B-lineage cells expressed the CB3 antigenon their surface (Fig. 4c). Among the CD19+ population, allof the CB3+ cells expressed ,u heavy chains but the CB3-cells did not (Fig. 4d). Moreover, the intensity ofCB3 antigenexpression correlated in linear fashion with that of the ,uheavy chains, as has been shown for the B29 gene product inmurine B cell lines (19). Surface expression of the CB3

207 697 Ramos IM9

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FIG. 3. Cell surface expression of the CB3-1 antigen and slg onpre-B and B cell lines. The 207 (p-) and 697 (A+) pre-B cells and theRamos [IgM(A)+] and IM9 [IgGl(K)+] B cells were incubated withpurified CB3-1 and anti-human ,A mAb SA-DA4-4 (207, 697, andRamos) or anti-human K mAb 187-1 (IM9), followed by biotinylatedgoat anti-mouse Ig antibody and phycoerythrin-conjugated strepta-vidin. Isotype-matched control antibodies served as negative con-trols (dotted lines).

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FIG. 4. Cell surface expression ofthe CB3-i antigen during B-celldifferentiation. (a) Mononuclear cells from normal peripheral blood(peripheral blood lymphocytes, PBL) were differentially stained withthe CB3-1 and anti-CD19 mAbs. (b) Bone marrow mononuclear cellsfrom a myeloma patient were stained with CB3-1 and anti-CD38antibodies. Large cells corresponding to myeloma plasma cells weregated on the basis of light-scatter characteristics for analysis of theirCB3-1 and CD38 expression. (c and d) Mononuclear cells from afetalbone marrow sample were differentially stained with CB3-1, goatanti-human 1t antibodies, and anti-CD19 mAb. Lymphoid cells weregated for immunofluorescence analysis of expression of the CB3-1antigen and CD19 (c). The CD19+ B-lineage cells were gated foranalysis of the expression of the CB3-1 antigen and surface ,u (d).

antigen thus begins with surface ,u heavy-chain expression,continues throughout B-cell maturation and isotype switch-ing, and terminates with plasma-cell differentiation.

Cytoplasmic Expression of the CB3 Antigen During B-CellDifferentiation. Next, we examined the cytoplasmic expres-sion ofthe CB3 antigen in pro-B (,- TdT+), pre-B (,u + TdT-),and B cells (sIgM+) in four fetal bone marrow samples. Morethan 80o of the u- TdT+ pro-B cells contained detectableamounts of the CB3 antigen in their cytoplasm. In keepingwith this observation, 26% of the cytoplasmic CB3+ lympho-cytes in fetal bone marrow were ,u-, whereas all of the ,u+pre-B and B cells contained the CB3 antigen. Interestingly,while most myeloma plasma cells did not appear to expressthe antigen, CB3 immunofluorescence could be visualized inthe Golgi region of a subpopulation of myeloma cells. Weconclude that expression of the CB3 antigen begins withnuclear TdT expression in human pro-B cells and persistsuntil the onset of terminal plasma-cell differentiation.

Molecular Heterogeneity of Ig-Associated Molecules DuringB-Cell Differentiation. When normal B cells were lysed in therelatively mild digitonin detergent, both the anti-light chainmAbs and CB3 mAbs precipitated Aand 8 heavy chains, lightchains, and the Ig-associated molecules (Fig. 5b, lanes 3 and6), indicating that sIgM and sIgD were physically associatedwith the CB3 antigen complex in normal B cells. The 'y heavychains could not be identified unambiguously in digitoninlysates because of the overlapping p45 band. Another bandbetween the 8 and y heavy chains, the identity of which isuncertain (marked X in Fig. 5a, lane 6), was observed inB-cell lysates. The X protein was prominant in both anti-lightchain and CB3 immunoprecipitates, but not in anti-heavychain immunoprecipitates.

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FIG. 5. Molecular heterogeneity ofIg-associated molecules observed as a function of B-cell differentiation and isotype differentiation. RamosB cells, normal B cells from peripheral blood, and 697 pre-B cells were labeled with 125I and lysed with either NP-40 (a and c) or digitonin (band d). Lysates were incubated with Sepharose 4B beads coupled with anti-s mAb (lanes 1 and 4), a mixture of anti-K plus anti-A mAbs (lane3), anti-8 mAb (lane 5), or CB3-1 (lanes 2 and 6) and immunoprecipitates were analyzed by SDS/1Oo PAGE under reducing conditions. Bandscorresponding to At, 8, and y heavy chains, light chains (L), surrogate light chains (*L), and an unidentified protein (X) are indicated byarrowheads and each Ig-associated molecule is indicated by arrows. A faint 53-kDa band in lane 5 of b is indicated by an arrow.

Interestingly, differences were evident in the characteris-tics of molecules associated with the tL and 8 chains. First, arelative paucity of sIgD-associated molecules was observedin comparison with the relative abundance of sIgM-associated molecules (Fig. 5b, lanes 4 and 5). This relativedeficit was especially notable for the p45 a-chain component,instead of which a faint protein band of 53 kDa was found tobe associated with sIgD (Fig. 5b, lane 5, arrow). In addition,the sIgM-associated p37 (3-chain protein identified by theCB3 antibodies (Fig. 2) was slightly larger than the compa-rable sIgD-associated band, which was -36 kDa (Fig. 5b,lanes 4 and 5).From NP-40 lysates of peripheral B cells, anti-light chain

mAbs precipitated heavy chains together with light chains,whereas the CB3 antibodies precipitated broad p45 and p37bands (Fig. 5a, lanes 3 and 6). Unexpectedly, under theserelatively harsh detergent conditions some 1L heavy chainswere also observed in the CB3 immunoprecipitates (Fig. 5a,lane 6). The relative intensities of the heavy-chain bands inanti-light chain immunoprecipitates were 8 > IL (Fig. 5a, lane3), whereas the relative intensities in the CB3-1 immunopre-cipitates were IL >> 8 (Fig. 5a, lane 6), indicating consider-able variability in the strength of association between thedifferent sIg isotypes and the a and 8 chains.The CB3 immunoprecipitates from NP-40 and digitonin

lysates of the 697 pre-B cell line also differed in severalrespects (Fig. 5 c and d). First, in comparison with the anti-Pantibody, the CB3 mAbs were relatively inefficient in pre-cipitation ofthe solubilized receptor units present in digitoninlysates. Second, the p34 j-chain candidate identified by theCB3 mAbs in Western blots of pre-B cells (Fig. 2) wasespecially prominent in NP-40 lysates. In addition, the p40and p45 a-chain species noted in B cells were largely replacedby 37- and 48-kDa proteins in the 697 pre-B cells. Thesedifferences, which were also observed in other pre-B (Nalm6,SMS-SB, BLIN-1) and B (Daudi) cell lines, reveal an inter-esting pattern of molecular heterogeneity in the slg-associated a and 8 chains during the progressive differenti-ation of pre-B cells to isotype-switched B cells. The apparent

molecular mass of a and chains from both pre-B and B cellswas reduced to -28 kDa following removal of N-linkedoligosaccharides by N-Glycanase treatment.

DISCUSSIONSeveral lines of evidence indicate that the CB3-i and -2antibodies recognize an external epitope on one of theIg-associated molecules. First, from NP-40 detergent lysatesof B cells, the CB3 antibodies precipitated a complex ofproteins distinct from the Ig heavy and light chains, whereasfrom B-cell lysates obtained with a milder detergent, digito-nin, these antibodies precipitated the same non-Ig complextogether with Ig heavy and light chains, although neitherantibody reacted with secreted IgM in a sensitive immunoen-zymatic assay. Of the three prominent disulfide-linked pro-teins, p45, p40, and p37, that were noncovalently associatedwith membrane-bound IgM on the B-cell lines, the CB3antibodies recognized p37 together with a minor p34 molec-ular species. Since mAbs raised against peptides of themb-l-encoded a chains recognize the p45 and p40 molecules(18), our data suggest that the CB3 antibodies recognize the(3chain products of the human B29 gene equivalent. Theidentification and characterization of the B29 gene counter-part in humans will allow formal testing of this interpretation.

Transcriptional activity ofthe mouse B29 (3-chain gene andofboth mouse and human mb-i a-chain genes can be detectedin pro-B cells, in which Ig heavy-chain genes are in germ-lineconfiguration, and in all types ofB cells, but not in terminallydifferentiated plasma cells (1, 2, 17). In accordance with theseresults, we observed that most TdT1 pro-B cells and all oftheIL+ pre-B cells expressed the CB3 antigen in their cytoplasm,whereas most myeloma plasma cells did not. Since nuclearTdT is expressed before B cells productively rearrange theirIg heavy-chain genes (20), this result indicates the onset of(3-chain expression at roughly the same stage in differentia-tion. However, surface expression of the CB3 (chain anti-gen was restricted to lymphocytes that expressed either the

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,u heavy chain/surrogate light chain complex or conventionalsIg isotypes.An interesting pattern of molecular heterogeneity of the

human Ig-associated molecules emerged in our studies ofpre-B and B cells. (i) The data indicate size variations in bothtypes of Ig-associated molecules as a function of maturationalstage. While the putative a- and (3-chain species associatedwith the p/surrogate light chain receptors on pre-B cell lineswere largely of 48 and 34 kDa, respectively, those associatedwith IgM on B cells were primarily 45 and 37 kDa in apparentmolecular mass. (ii) Size differences in both types of Ig-associated chains were observed for the sIgM and sIgDreceptors on normal B cells. While a- and (3-chain species of53 and 36 kDa were associated with IgD, proteins of 45 and37 kDa were associated with IgM receptors. Differentlyglycosylated forms apparently account for this size hetero-geneity, since the same protein core size (-28 kDa) wasobserved for a and ( chains from both pre-B and B cells.These results complement and extend data obtained in stud-ies of mice, where only the mb-i a-chain gene productsassociated with IgM and IgD differ in apparent size due todifferential glycosylation (5, 21). (iii) We observed an appar-ent variation between sIgM and sIgD in the strength of theirassociation with their respective a- and (-chain componentson normal B cells. In the digitonin lysates, the CB3 antibodiesprecipitated ji and 8 heavy chains together with a and 83chains. The CB3 antibodies also coprecipitated ,u, but not 8,heavy chains from NP-40 lysates. This is in spite of the factthat analysis of sIg isotype distribution revealed highersurface levels of 8 than ,u heavy chains on the normal B-cellpopulation. This relatively weak association ofboth the a and,B chains with sIgD on human B cells complements the resultsof experiments in mice, where gene transfection studies haveindicated that sIgD expression may occur without either a orP chains (7, 22). In the latter case, however, the IgD thatreached the surface of cells lacking in a and ( chains provedto be attached via glycosyl-phosphatidylinositol linkagerather than via the usual transmembrane domain (23). Inhumans, the transmembrane form of sIgD appears to pre-dominate, as its expression is resistant to treatment withphosphatidylinositol-specific phospholipase C (data notshown) and both a and P chains are abundant in B cellsbearing sIgD and sIgM.

Crosslinkage of sIgM molecules on mature B cells induceshydrolysis of phosphatidylinositol bisphosphate leading tocell proliferation (24, 25). Signaling through sIgM on imma-ture B cells instead induces growth inhibition through acti-vation of a protein-tyrosine kinase pathway (26-28), eventhough these cells have an intact phosphatidylinositol path-way that can be otherwise activated (12). Signaling throughIgM and IgD receptors on the same B cell may also result indifferent biological effects (12, 13). Therefore, it is attractiveto speculate that the heterogeneity of the sIg-associatedmolecules that we have observed may be an importantvariable in signal transduction via the different slg receptorunits expressed as a function ofpre-B- and B-cell maturation.The CB3 mAbs provide another surface marker for B cells

and should be helpful in further delineation of the signaltransduction pathways activated via antibody receptors dur-ing different stages in B-cell differentiation. The CB3 anti-bodies could also be of therapeutic value, since they reactwith all sIg+ B cells and can down-modulate antibody recep-tors in a fashion comparable to the modulation of T-cell

receptors by anti-CD3 antibodies, which have proven valu-able for the diagnostic and therapeutic manipulation ofT cells(29).

We thank Drs. Cezar Nunez and Linda Billips for help in preparingbone marrow samples. This work was supported in part by GrantsA130879, A118745, and CA13148 awarded by the National Institutesof Health. M.D.C. is an Investigator of the Howard Hughes MedicalInstitute.

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